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| United States Patent |
5,753,451
|
|
Smith
|
May 19, 1998
|
Process for determining specific gravity of adulterants in urine
employing an automatic analyzer
Abstract
An automatic analyzer is used to determine out of range specific gravity of
adulterants in a urine sample. An aliquot of the urine is mixed with a
buffer and ion detector and thereafter with a polymer activator for ion
detection. The mixture is analyzed by setting a spectrophotometer in the
automatic analyzer at about 600 nanometers, setting the calibrating value
for specific gravity at 1.000 and 1.0500 and reading a color change to
determine the presence of adulterants.
| Inventors:
|
Smith; Jack V. (St. Petersburg, FL)
|
| Assignee:
|
Chimera Research & Chemical, Inc. (Tampa, FL)
|
| Appl. No.:
|
786118 |
| Filed:
|
January 17, 1997 |
| Current U.S. Class: |
435/12; 73/32R; 422/56 |
| Intern'l Class: |
C12Q 001/58 |
| Field of Search: |
435/12
422/56
436/2,24,164
73/32 R
|
References Cited
U.S. Patent Documents
| 4318709 | Mar., 1982 | Falb et al. | 23/230.
|
| 4376827 | Mar., 1983 | Stiso et al. | 436/2.
|
| 4590800 | May., 1986 | Shimoda | 73/449.
|
| 5179027 | Jan., 1993 | Fisher | 436/56.
|
| 5302531 | Apr., 1994 | Bauer | 436/74.
|
| 5320969 | Jun., 1994 | Bauer et al. | 436/84.
|
| 5350694 | Sep., 1994 | Zimmerle | 436/2.
|
Other References
Mikkelsen S., Identification of Substances Interfering With Illicit Drug
Testing, AFIT/CI/NR-88-116 Masters Thesis, 1988.
|
Primary Examiner: Gitomer; Ralph
Attorney, Agent or Firm: Larson & Larson, P.A., Larson; Herbert W.
Parent Case Text
PRIOR APPLICATIONS
This application is a continuation from Ser. No. 08/497,641, filed Jun. 30,
1995, which is a continuation-in-part from application Ser. No.
08/276,502, filed Jul. 18, 1994, which is a continuation from Ser. No.
07/700,713, filed May 16, 1991, all now abandoned.
Claims
I claim:
1. A process employing an automatic analyzer to determine specific gravity
outside of normal range on a urine sample, comprising:
(a) mixing an aliquot of the urine sample with a divalent buffer in an
aqueous solution containing at lest 0.1% by weight surfactants to form a
first mixture,
(b) thereafter, mixing the first mixture with methyl vinyl ether/maleic
anhydride, at least 0.1% by weight surfactants and a color indicator to
form a second mixture,
(c) placing the second mixture into a cuvette within the automatic
analyzer,
(c) setting a spectrophotometer in the automatic analyzer for reading at
about 600 nanometers,
(e) setting a calibrating value for specific gravity in the automatic
analyzer between 1.000 and 1.0500, and
(f) reading a color change to determine a presence of an out of normal
range specific gravity below 1.005 or above 1.030 in the urine sample with
a sensitivity of .+-. 0.001.
2. The process according to claim 1 wherein the first mixture is obtained
by mixing with the urine, an aqueous solution of potassium phosphate,
sodium phosphate, sodium azide, and the surfactant octoxynol.
3. the process according to claim 1 wherein at least one surfactant added
to the first mixture is polyoxyethylene 23 lauryl ether.
4. The process according to claim 1 wherein the pH of the first mixture is
adjusted to between 6.85 and 6.90.
5. The process according to claim 1 wherein isopropanol and water are added
to the second mixture in step (b).
6. The process according to claim 5 wherein the color indicator is
bromethymol blue and a surfactant added is polyoxyethylene 23 lauryl
ether.
7. The method of quantitatively determining out of normal range specific
gravity of a urine sample comprising:
(a) mixing the urine sample with a divalent buffer in an aqueous solution
containing at least 0.1% by weight surfactants to form a first mixture,
(b) mixing the first mixture with methyl vinyl ether/maleic anhydride, at
least 0.1% by weight surfactants and a color indicator to form a second
mixture,
(c) placing the second mixture into a cuvette within an automatic analyzer,
(d) setting a spectrophotometer in the automatic analyzer for reading at
about 600 nanometers,
(e) setting a calibrating value for specific gravity in the automatic
analyzer between 1.000 and 1.0500, and
(f) reading a color change to quantitatively determine a presence or
absence of an out of normal range specific gravity below 1.005 or above
1.030 in the urine sample with a sensitivity of .+-.0.001.
Description
BACKGROUND OF THE INVENTION
This invention relates to a single reagent system for use in determining
the specific gravity in urine, or other fluids, being screened for drugs
of abuse. This invention is particularly useful in automated analyzers
used in screening for drugs of abuse.
As the use of illicit drugs in the workplace, public transportation,
professional and amateur athletics and the like has grown, public concern
for the health and safety of individuals, as well as concern for the
negative impact of such drug use on productivity of industry, and its
inherent economic impact, and the general well being and health of the
community at large has grown as well. Such concern has led to the use of
analysis of urine as a way to detect and deter drug use. Such testing for
drugs of abuse in industry, as for prospective and current employees,
military personnel, transportation employees, professional and amateur
atheletes, as well as people under supervision of the criminal justice
system, has become a relative common occurence.
Because of the intrusive nature of such testing, commonly performed by
examining a urine sample, the testing procedure must withstand vigorous
scrutiny. As a positive test result of screening for drugs of abuse may
have serious impact on the life of a person being tested, the incentive
for the drug user to alter the test specimen is high. The users of drugs
of abuse have developed a number of ways to adulterate the collected
specimen, thus attempting to produce a false negative result in the drug
screening test being conducted.
A user of drugs of abuse may attempt to affect the test results, thus
producing a false negative test result, or upon occasion, a false positive
result, as by: a) dilution--efforts to reduce the drug concentration in
the urine sample; b) substitution--substitution of liquids such as clean
(that is, drug-free) urine, soda, tea, apple juice for the drug-containing
sample; or c) adulteration--addition to the urine specimen of foreign
material in an attempt to invalidate the test.
Illicit drug users have learned to falsify urine screen tests by in vitro
adulteration of the urine sample by the addition of several readily
available agents, including household products, among others, NaCl, soap,
such as hand or dish soap, bleach, vinegar, Drano Pipe Cleaner,
NaHCO.sub.3, Visine eye drops, GOLD SEAL TEA (available in natural food
stores), or H.sub.2 O.sub.2.
Additionally, users of drugs of abuse may eliminate some drugs more rapidly
from their bodies by altering their urinary pH. Abusers of phencyclidine
or amphetamines may be treated with NH.sub.4 Cl to hasten detoxification,
thus increasing the rate at which substances (phencyclidine or
amphetamines) are eliminated from their bodies. This treatment with
NH.sub.4 Cl also results in lowering the pH of the user's urine.
While the use of some in vitro adulterants can be eliminated by the direct
observation of the test subject during the collection process, such direct
observation is often deemed unacceptable. In vivo adulterants represent an
additional burden to the screening processor because they are consumed by
the drug user several hours or days prior to screening of the sample, and
can be detected only by laboratory means.
Such adulteration can affect all three commonly used methods for drugs of
abuse, namely: fluorescent polarization immunoassay (FPIA),
radioimmunoassay (RIA), and enzyme immunoassay (EMIT or EIA).
Consequently, clinical chemistry literature recommends that testing for
drugs of abuse in urine samples include testing for adulterants to
identify urine samples which have been adulterated. See Mikkelsen and Ash,
"Adulterants Causing False Negatives In Illicit Drug Testing", Clin. Chem.
34/11, 2333-2336 (1988); and Warner, "Interference of Common Household
Chemicals In Immunoassay Methods For Drugs Of Abuse", Clin. Chem. 35/4,
648-651 (1989).
Accordingly, a need exists for providing an easy and convenient manner by
which to make determination of the presence of adulterants in urine
samples which are being tested for drugs of abuse. A further need exists
for a convenient manner by which such determinations may be made in
conjunction with an automatic analyzing process for drugs of abuse.
SUMMARY OF THE INVENTION
The present invention relates to a single channel reagent to detect
simultaneously multiple levels of specific gravity in urine or other
fluids. This reagent is designed to be used on automated analyzers used
for drugs of abuse testing.
The purpose of the reagent is to facilitate the conducting of testing for
specific gravity simultaneously while conducting drug tests on the same
automatic analyzer. Specifically, if the specific gravity of the urine, or
other sample fluid being tested, is out of the normal range, that is,
greater than 1.030 specific gravity units or less than 1.005 specific
gravity units, such variation from the normal range will cause false
negative readings, when tested by a common drug-screening method, namely,
EIA (enzyme immunoassay) in testing for drugs of abuse, such as:
methadone, opiate, THC, barbituate, PCP, amphetamine, benzodiazepine,
cocaine, propoxyphene. For example, the adulteration of a urine sample
with a common agent such as table salt, NaCl, will result in a change in
the specific gravity of the urine sample.
It is helpful to note that upon occasion, abnormally high amounts of
urinary constituents, as excreted by the body, can cause an increase in
specific gravity values. Also, the disease of diabetes insipidus is
characterized by large urine volumes of low specific gravity, usually
between the values of 1.001 and 1.003.
Use of the reagent of this invention permits the technician conducting the
test to halt the testing process, or assay, as soon as the out-of-range
specific gravity determination is made. The ability to terminate the
screening process by ascertaining that the specific gravity of the sample
is out of range, and therefore presumably adulterated, would result in
reduced technician's efforts and time, providing an economic savings to
the testing laboratory. Furthermore, the early interruption and cessation
of the automated screening process may facilitate earlier obtention of a
substitute specimen from the person being tested, providing more accurate
determinations to the agency which had determined the original necessity
for the test.
The use of the instant reagent system permits the determination of the
specific gravity of the testing sample to be done by the automated
substance abuse testing program, rather than the relatively cumbersome
methods of hand-held methods of testing for adulteration, such as the use
of pH test (litmus) paper, which must be dipped in the urine, by pH
metering or other manual methods.
The instant reagent system comprises an aqueous solution of different
specific gravity indicators that effect a color change, depending on the
specific gravity of the urine, or other sample fluid being tested. The
reagent is based on an indicator principal (redox) which gives a broad
range of colors throughout the urinary specific gravity range, normally in
the range of 1.005 to 1.030, but usually remaining between 1.010 and
1.025. A sample of urine is mixed with the reagent in a specific ratio,
and the mixture of urine and reagent will exhibit a color change,
depending on the specific gravity of the urine. The color change results
in a change in the light absorbance, which may be detected by a
spectrophotometer at 600 nanometers.
DETAILED DESCRIPTION OF THE INVENTION
The specific gravity reagent of the instant invention comprises an aqueous
solution of a divalent buffer, which is mixed with the urine or other
fluid sample to be tested. The mixture of the fluid sample with the
divalent buffer, which serves as a reaction stabilizer, and which also
includes an activator, or ion detector, hereinafter referred to as R1
Diluent, is then mixed with a second aqueous solution, referred to
hereinafter as R2 Color, which includes a particular polymer, which
functions as an activator for ion detection, and color indicator. The
entire mixture is then inserted into the instrument, and the absorbance
read for comparison against known standards.
The formulations for R1 Diluent and R2 Color are prepared as follows:
R1 DILUENT is prepared as follows:
10.0 g sodium thiosulfate
100 ml isopropanol
6.8 g potassium phosphate monobasic (KH.sub.2 PO.sub.4)
7.1 g sodium phosphate dibasic 12-hydrate Na.sub.2 PO.sub.4)
0.1 g sodium azide
1.0 ml Triton X-100 octoxynol
1.0 ml Brij 35 solution, 30% w/v (polyoxyethylene 23 lauryl ether)
The ingredients are mixed together, and the pH of the solution is adjusted
to between 6.85-6.90 with sodium hydroxide or lactic acid, as appropriate.
The solution is then brought to 1.1 liter with reagent grade distilled
water.
R2 COLOR
A solution, known hereinafter as R2 COLOR CONCENTRATE, is prepared
according to the following formulation:
30.0 g methyl vinyl ether/maleic anhydride
750 ml isopropanol
250 ml Reagent grade distilled water.
The solution is mixed thoroughly, for 48 to 72 hours, or until compound
goes into solution.
R2 COLOR is prepared as follows:
175 ml R2 COLOR CONCENTRATE
1.5 g Bromthymol Blue (or other indicator as indicated in list A below, or
mixture of color indicators)
6.0 ml Brij 35 solution, 30% w/v (polyoxyethylene 23 lauryl ether)
1.0% Isopropanol
The pH of the solution is adjusted to 8.0 with sodium hydroxide or lactic
acid, as appropriate. The R2COLOR solution is brought to a quantity of 6.0
L with reagent grade distilled water.
A - Indicators for R2COLOR
1.5 g Bromthymol Blue
1.5 g alizarin yellow R
1.5 g Tetrabromphenol Blue
1.5 g Brom-chlorphenol Blue
1.5 g Thymol Blue
1.5 g Metacresol Purple
1.5 g Phenolthalein
1.5 g thymolphthalein
1.5 g o-cresolphthalein
1.5 g tropaeolin 000 no. 1
1.5 g cresol red
1.5 g neutral red
1.5 g p-nitrophenol
1.5 g dibromophenol-tetrabromophenol-sulfonphthalein
1.5 g bromphenol red
1.5 g bromcresol purple
1.5 g azolitmin
1.5 g benzoyl auramine G
1.5 g chlorphenol red
1.5 g litmus
1.5 g lacmoid
1.5 g methyl red
1.5 g 2,5 - dinitrophenol
1.5 g 2,4 - dinitrophenol
1.5 g bromalsol green
1.5 g sodium alizarinsulfonate
1.5 g napthyl red
1.5 g p-ethoxchrysoidine
1.5 g methyl orange
1.5 g congo red
1.5 g bromphenol blue
1.5 g methyl yellow
1.5 g quinaldine red
1.5 g tropaelin 00
1.5 g p-xylenol blue
1.5 g metanil yellow
1.5 g methyl violet
(Note that the method of preparation of the R2COLOR solution is the same,
with any of the indicators in list A.)
The reagent system of the instant invention is intended for use on
automatic analyzers, such as enzyme immunoassay analyzers (EMIT), such as
Olympus AU 5000 series, Monarch, Hitachi 700 series, among others. On
these instruments, the reagent is used in the following manner: 5 .mu.L of
urine sample is placed in a sample tube and mixed with 50 .mu.L of R1
DILUENT. This mixture is then mixed with 300 .mu.L of R2 COLOR. The
instrument spectrophotometer is set at 600 nm, and the calibrator values
of the instrument are set at a normal high value of 1.0300 and the normal
low value set at 1.0050. The absorbance of the sample is then measured.
In the instant invention, when urine which has been adulterated, resulting
in a change in the specific gravity, is mixed with the reagent system in
the prescribed ratio, the indicator will cause the sample mixture to
change color, depending on the specific gravity (ionic strength) of the
solution. Such indication may be seen in a manual inspection, but is
especially intended for use in automatic analysis, such as those which
employ spectophotometric means of inspection.
Specifications for running the urine samples through three specific
instruments, of the enzyme immunoassay type (EMIT) Olympus, Hitachi and
Monarch, are listed below. The settings are intended as guidelines, and
are set forth with the understanding that those skilled in the art would
recognize that such parameters will vary from instrument to instrument.
The suggested specifications are as follows:
Parameter Settings for Olympus AU5000
1. Turn on yes/no--yes
Sample vol.: 05 .mu.L
R1 DILUENT vol.: 50 .mu.L
R2 COLOR vol.: 300 .mu.L
2. Activate--W3
R1--yes
R2--yes
Wavelength 1: 600 nm.
Wavelength 2: 000 nm
Curve (slope): --
Measuring points (photo station)
start at 0
end at 3
11. Normal High Value-1.0300
Normal Low Value-1.0050
12. O.D. range-2.5000 to -2.0000
Calibration method (TYPE): AA
1) Concentration 1=1.005
2) Concentration 2=1.030
Note: Group Number (#) Blank (to be determined by testing facility), Cal 1
concentration--1.005Group Number (#) Blank, Cal 2
______________________________________
Parameter Settings for Monarch
______________________________________
Identification Parameters:
Test code 116
Test name SG
Test mnemonic SG
Optical mode Absorbance
Response algorithm Final point
Result algorithm Linear
Loading Parameters:
Loading type Load
Analyze
Reagent blank Off
Reference type Diluent
Calibrator type Test specific
Sample volume 4 .mu.L
Sample diluent 5 .mu.L
Reagent diluent 10 .mu.L
1st reagent 175 .mu.L
2nd reagent 40 .mu.L
1st rgt bar code 1 k
Data Acquisition Parameters:
Analysis type Mix run
Temperature 25 C
Delay time 30 sec.
Interval time 30 sec
No. of data points 1
Filter 1 600 nm
Filter 2 600 nm
Monochromator 1 600 nm
Monochromator 2 600 nm
Compatibility None
Data Integrity Parameters:
Integrity tests Normal range
Lower limit 1.005
Upper limit 1.030
Data Fit Parameters:
Calibrator 1 1.005
Calibrator 2 1.030
Correction mode none
Units none
No. of decimal places 4
Calculated Parameters:
Data edited (date)
Time edited (time)
Not calibrated run time
30 sec.
______________________________________
It is recommended that calibrator values for the automated analyzers be set
at values of 1.005 and 1.030, with the instrument set to flag values at or
below 1.003, as well as values at or above 1.035.
Example
Sixty urine specimens were assayed for specific gravity using the reagent
system of the invention on the Hitachi 717 by an independent laboratory.
The assay was compared to the reference assay (refractometer) using
correlation and regression analysis, along with estimated standard of
error of the regression. Also the Ames Dipstick method was used with the
reference method. The assay was conducted in conjunction with an assay to
evaluate automated pH screening, using pH reagents of the inventor of the
instant specific gravity reagent system.
The specific gravity reagent of the instant invention was used, per
manufacturer's instructions in the Hitachi assay.
The laboratory used the BIGRESS program (Bivarate Regression) on the
EXPLORE (statistical software package) to conduct the statistical analysis
on the results.
The specific gravity values for 60 urine samples measured by the
Hitachi/reagent method, the reference/refractometer method and the Ames
dipstick method are as follows:
______________________________________
Hitachi Reference
Dipstick
Sample # Value Value Value
______________________________________
1 1.004 1.013 1.020
2 1.010 1.011 1.020
3 1.031 1.028 1.030
4 1.014 1.020 1.020
5 1.022 1.033 1.030
6 1.017 1.023 1.030
7 1.013 1.017 1.020
8 1.004 1.008 1.010
9 1.009 1.018 1.030
10 1.013 1.021 1.030
11 1.007 1.022 1.020
12 1.015 1.021 1.030
13 1.018 1.020 1.030
14 1.017 1.030 1.030
15 1.006 1.013 1.020
16 1.019 1.024 1.030
17 1.021 1.025 1.030
18 1.011 1.016 1.020
19 1.005 1.006 1.020
20 1.009 1.010 1.030
21 1.011 1.016 1.030
22 1.019 1.024 1.020
23 1.009 1.016 1.020
24 1.005 1.011 1.015
25 1.006 1.005 1.015
26 0.997 1.017 1.020
27 1.006 1.013 1.020
28 1.006 1.008 1.030
29 1.012 1.026 1.030
30 1.016 1.020 1.020
31 1.011 1.031 1.030
32 1.020 1.028 1.030
33 1.027 1.028 1.025
34 1.016 1.019 1.030
35 1.021 1.023 1.030
36 1.022 1.019 1.020
37 1.008 1.010 1.030
38 1.020 1.018 1.025
39 1.007 1.013 1.025
40 1.012 1.018 1.030
41 1.002 1.022 1.020
42 1.022 1.024 1.030
43 1.027 1.027 1.030
44 1.029 1.024 1.030
45 1.012 1.020 1.020
46 1.006 1.019 1.030
47 1.021 1.024 1.020
48 1.003 1.015 1.025
49 1.002 1.017 1.025
50 1.000 1.017 1.025
51 1.007 1.012 1.025
52 1.023 1.024 1.025
53 1.012 1.023 1.030
54 1.026 1.030 1.030
55 1.013 1.017 1.030
56 1.012 1.012 1.030
57 1.014 1.016 1.030
58 1.014 1.015 1.030
59 1.005 1.007 1.025
60 1.016 1.022 1.030
______________________________________
After correlating the data, 3% (or 2 out of 60 specimens) had values less
than 1.003, suggesting confirmation of the results with a refractometer.
Using the above-noted software for the statistical analysis, laboratory
noted that the mean results of the Hitachi method, using the instant
reagent, would not vary more than +/-.0014, suggesting a very high level
of reliabilty.
It will be understood that the embodiments described in the instant
application are merely exemplary and that a person skilled in the art may
make variations and modifications without departing from the spirit and
scope of the invention. All such modifications and variations are to be
included within the scope of the invention as defined in the appended
claims:
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